Mark T. Quinn

Superoxide Production and Expression of Nox Family Proteins in Human Atherosclerosis

Background—NAD(P)H oxidases are important sources of superoxide in the vasculature, the activity of which is associated with risk factors for human atherosclerosis. This study was designed to investigate the localization of superoxide production and the expression of the Nox family of NAD(P)H oxidase proteins (gp91phox, Nox1, and Nox4) in nonatherosclerotic and atherosclerotic human coronary arteries. Methods and Results—In coronary artery segments from explanted human hearts, we examined intracellular superoxide production with dihydroethidium. In nonatherosclerotic coronary arteries, superoxide was present homogenously throughout the intima, media, and adventitia. In atherosclerotic arteries, there was an additional intense area of superoxide in the plaque shoulder, which is rich in macrophages and &agr;-actin–positive cells. p22phox colocalized with gp91phox mainly in macrophages, whereas Nox4 was found only in nonphagocytic vascular cells. Expression of gp91phox and p22phox mRNA was associated with the severity of atherosclerosis. gp91phox correlated with the plaque macrophage content, whereas Nox4 correlated with the content of &agr;-actin–positive cells. Nox1 expression was low both in human coronary arteries and isolated vascular cells. Conclusions—Several Nox proteins, including gp91phox and Nox4, may contribute to increased intracellular oxidative stress in human coronary atherosclerosis in a cell-specific manner and thus may be involved in the genesis and progression of human coronary atherosclerotic disease.

Expression of a Functionally Active gp91phox-Containing Neutrophil-Type NAD(P)H Oxidase in Smooth Muscle Cells From Human Resistance Arteries Regulation by Angiotensin II

A major source of vascular smooth muscle cell (VSMC) superoxide is NAD(P)H oxidase. However, the molecular characteristics and regulation of this enzyme are unclear. We investigated whether VSMCs from human resistance arteries (HVSMCs) possess a functionally active, angiotensin II (Ang II)–regulated NAD(P)H oxidase that contains neutrophil oxidase subunits, including p22phox, gp91phox, p40phox, p47phox, and p67phox. mRNA expression of gp91phox homologues, nox1 and nox4, was also assessed in HVSMCs, human aortic smooth muscle cells, and rat VSMCs. HVSMCs were obtained from resistance arteries from gluteal biopsies of healthy subjects. gp91phox and nox4, but not nox1, were detected in HVSMCs. Nox1 and nox4, but not gp91phox, were expressed in human aortic smooth muscle cells and rat VSMCs. All NAD(P)H oxidase subunits were present in HVSMCs as detected by reverse transcriptase–polymerase chain reaction and immunoblotting. Ang II increased NAD(P)H oxidase subunit abundance. These effects were inhibited by cycloheximide. Acute Ang II stimulation (10 to 15 minutes) increased p47phox serine phosphorylation and induced p47phox and p67phox translocation. This was associated with NAD(P)H oxidase activation. In cells transfected with gp91phox antisense oligonucleotides, Ang II–mediated actions were abrogated. NADPH-induced superoxide generation was reduced by gp91ds-tat and apocynin, inhibitors of p47phox-gp91phox interactions. Our results suggest that HVSMCs possess a functionally active gp91phox-containing neutrophil-like NAD(P)H oxidase. Ang II regulates the enzyme by inducing phosphorylation of p47phox, translocation of cytosolic subunits, and de novo protein synthesis. These novel findings provide insight into the molecular regulation of NAD(P)H oxidase by Ang II in HVSMCs. Furthermore, we identify differences in gp91phox homologue expression in VSMCs from rats and human small and large arteries.

Distinct Subcellular Localizations of Nox1 and Nox4 in Vascular Smooth Muscle Cells

Objective—Reactive oxygen species (ROS) that act as signaling molecules in vascular smooth muscle cells (VSMC) and contribute to growth, hypertrophy, and migration in atherogenesis are produced by multi-subunit NAD(P)H oxidases. Nox1 and Nox4, two homologues to the phagocytic NAD(P)H subunit gp91phox, both generate ROS in VSMC but differ in their response to growth factors. We hypothesize that the opposing functions of Nox1 and Nox4 are reflected in their differential subcellular locations. Methods and Results—We used immunofluorescence to visualize the NAD(P)H subunits Nox1, Nox4, and p22phox in cultured rat and human VSMC. Optical sectioning using confocal microscopy showed that Nox1 is co-localized with caveolin in punctate patches on the surface and along the cellular margins, whereas Nox4 is co-localized with vinculin in focal adhesions. These immunocytochemical distributions are supported by membrane fractionation experiments. Interestingly, p22phox, a membrane subunit that interacts with the Nox proteins, is found in surface labeling and in focal adhesions in patterns similar to Nox1 and Nox4, respectively. Conclusions—The differential roles of Nox1 and Nox4 in VSMC may be correlated with their differential compartmentalization in specific signaling domains in the membrane and focal adhesions.

Aldosterone-induced inflammation in the rat heart: Role of oxidative stress

Heart failure and hypertension have each been linked to an induction of oxidative stress transduced by neurohormones, such as angiotensin II and catecholamines. Herein, we hypothesized that aldosterone (ALDO) likewise induces oxidative stress and accounts for a proinflammatory/fibrogenic phenotype that appears at vascular and nonvascular sites of injury found in both right and left ventricles in response to ALDO/salt treatment and that would be sustained with chronic treatment. Uninephrectomized rats received ALDO (0.75 micro g/hour) together with 1% dietary NaCl, for 3, 4, or 5 weeks. Other groups received this regimen in combination with an ALDO receptor antagonist, spironolactone (200 mg/kg p.o. daily), or an antioxidant, either pyrrolidine dithiocarbamate (PDTC) (200 mg/kg s.c. daily) or N-acetylcysteine (NAC) (200 mg/kg i.p. daily). Unoperated and untreated age- and gender-matched rats served as controls. We monitored spatial and temporal responses in molecular and cellular events using serial, coronal sections of right and left ventricles. Our studies included: assessment of systolic blood pressure; immunohistochemical detection of NADPH oxidase expression and activity; analysis of redox-sensitive nuclear factor-kappaB activation; in situ localization of intercellular adhesion molecule-1, monocyte chemoattractant protein-1, and tumor necrosis factor-alpha mRNA expression; monitoring cell growth and infiltration of macrophages and T cells; and analysis of the appearance and quantity of fibrous tissue accumulation. At week 3 of ALDO/salt treatment and comparable to controls, there was no evidence of oxidative stress or pathological findings in the heart. However, at weeks 4 and 5 of treatment, increased gp91(phox) and 3-nitrotyrosine expression and persistent activation of RelA were found in endothelial cells and inflammatory cells that appeared in the perivascular space of intramural coronary arteries and at sites of lost cardiomyocytes in both ventricles. Coincident in time and space with these events was increased mRNA expression of intercellular adhesion molecule-1, monocyte chemoattractant protein-1, and tumor necrosis factor-alpha. Macrophages, lymphocytes, and proliferating endothelial and vascular smooth muscle cells and fibroblast-like cells were seen at each of these sites, together with an accumulation of fibrillar collagen, or fibrosis, as evidenced by a significant increase in ventricular collagen volume fraction. Co-treatment with spironolactone, PDTC, or NAC attenuated these molecular and cellular responses as well as the appearance of fibrosis at vascular and nonvascular sites of injury. Furthermore, elevated systolic blood pressure in ALDO-treated rats was partially suppressed by spironolactone or either antioxidant. Thus, chronic ALDO/salt treatment is accompanied by a time-dependent sustained activation of NADPH oxidase with 3-nitrotyrosine generation and nuclear factor-kappaB activation expressed by endothelial cells and inflammatory cells. This leads to a proinflammatory/fibrogenic phenotype involving vascular and nonvascular sites of injury found, respectively, in both normotensive and hypertensive right and left ventricles. Spionolactone, PDTC, and NAC each attenuated these responses suggesting ALDO/salt induction of oxidative/nitrosative stress is responsible for the appearance of this proinflammatory phenotype.

Assembly of the phagocyte NADPH oxidase: molecular interaction of oxidase proteins.

Phagocytes such as neutrophils play a key role in the bodys innate immune response to infection. These cells travel throughout the body in search of pathogens and are rapidly mobilized to sites of inflammation where they phagocytose these pathogens and subsequently release a variety of toxic oxygen radical species and proteolytic enzymes to directly destroy the engulfed particle. The generation of microbicidal oxidants by neutrophils results from the action of a multi‐protein enzymatic complex known as the NADPH oxidase. Altogether, there are currently seven proteins reported to be associated with the NADPH oxidase assembly. In resting neutrophils, these NADPH oxidase protein components are segregated into cytoplasmic and plasma membrane compartments. However, during assembly and activation of the NADPH oxidase, the cytosolic protein components translocate to the plasma membrane or phagosomal membrane where they assemble around a central membrane‐bound protein known as flavocytochrome b. This assembly process is highly regulated and involves multiple binding interactions between the individual NADPH oxidase proteins, resulting in an active oxidase complex. Over the past few years, a number of these sites of binding interaction between the oxidase proteins have been identified, leading to a clearer understanding of the intermolecular interactions occurring among protein components during the assembly process. In addition, this information has contributed to our understanding of the roles played by each protein during the activation and assembly process. In this review, we describe the key features of each NADPH oxidase protein and then summarize our current understanding of the specific molecular interactions occurring between these proteins, focusing on the role these protein:protein binding interactions play in the NADPH oxidase assembly process. J. Leukoc. Biol. 60: 677–691; 1996.

Novel Role of gp91phox-Containing NAD(P)H Oxidase in Vascular Endothelial Growth Factor–Induced Signaling and Angiogenesis

Vascular endothelial growth factor (VEGF) induces angiogenesis by stimulating endothelial cell proliferation and migration, primarily through the receptor tyrosine kinase VEGF receptor2 (Flk1/KDR). Reactive oxygen species (ROS) derived from NAD(P)H oxidase are critically important in many aspects of vascular cell regulation, and both the small GTPase Rac1 and gp91(phox) are critical components of the endothelial NAD(P)H oxidase complex. A role of NAD(P)H oxidase in VEGF-induced angiogenesis, however, has not been defined. In the present study, electron spin resonance spectroscopy is utilized to demonstrate that VEGF stimulates O2*- production, which is inhibited by the NAD(P)H oxidase inhibitor, diphenylene iodonium, as well as by overexpression of dominant-negative Rac1 (N17Rac1) and transfection of gp91(phox) antisense oligonucleotides in human umbilical vein endothelial cells (ECs). Antioxidants, including N-acetylcysteine (NAC), various NAD(P)H oxidase inhibitors, and N17Rac1 significantly attenuate not only VEGF-induced KDR tyrosine phosphorylation but also proliferation and migration of ECs. Importantly, these effects of VEGF are dramatically inhibited in cells transfected with gp91(phox) antisense oligonucleotides. By contrast, ROS are not involved in mediating these effects of sphingosine 1-phosphate (S1P) on ECs. Sponge implant assays demonstrate that VEGF-, but not S1P-, induced angiogenesis is significantly reduced in wild-type mice treated with NAC and in gp91(phox-/-) mice, suggesting that ROS derived from gp91(phox)-containing NAD(P)H oxidase play an important role in angiogenesis in vivo. These studies indicate that VEGF-induced endothelial cell signaling and angiogenesis is tightly controlled by the reduction/oxidation environment at the level of VEGF receptor and provide novel insights into the NAD(P)H oxidase as a potential therapeutic target for angiogenesis-dependent diseases.

Structure and regulation of the neutrophil respiratory burst oxidase: comparison with nonphagocyte oxidases

Neutrophils play an essential role in the body’s innate defense against pathogens and are one of the primary mediators of the inflammatory response. To defend the host, neutrophils use a wide range of microbicidal products, such as oxidants, microbicidal peptides, and lytic enzymes. The generation of microbicidal oxidants by neutrophils results from the activation of a multiprotein enzyme complex known as the reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, which is responsible for transferring electrons from NADPH to O2, resulting in the formation of superoxide anion. During oxidase activation, cytosolic oxidase proteins translocate to the phagosome or plasma membrane, where they assemble around a central membrane‐bound component known as flavocytochrome b. This process is highly regulated, involving phosphorylation, translocation, and multiple conformational changes. Originally, it was thought that the NADPH oxidase was restricted to phagocytes and used solely in host defense. However, recent studies indicate that similar NADPH oxidase systems are present in a wide variety of nonphagocytic cells. Although the nature of these nonphagocyte NADPH oxidases is still being defined, it is clear that they are functionally distinct from the phagocyte oxidases. It should be noted, however, that structural features of many nonphagocyte oxidase proteins do seem to be similar to those of their phagocyte counterparts. In this review, key structural and functional features of the neutrophil NADPH oxidase and its protein components are described, including a consideration of transcriptional and post‐translational regulatory features. Furthermore, relevant details about structural and functional features of various nonphagocyte oxidase proteins will be included for comparison.

AT1 Blockade Prevents Glucose-Induced Cardiac Dysfunction in Ventricular Myocytes Role of the AT1 Receptor and NADPH Oxidase

Abstract—Enhanced tissue angiotensin (Ang) II levels have been reported in diabetes and might lead to cardiac dysfunction through oxidative stress. This study examined the effect of blocking the Ang II type 1 (AT1) receptor on high glucose–induced cardiac contractile dysfunction. Rat ventricular myocytes were maintained in normal- (NG, 5.5 mmol/L) or high- (HG, 25.5 mmol/L) glucose medium for 24 hours. Mechanical and intracellular Ca2+ properties were assessed as peak shortening (PS), time to PS (TPS), time to 90% relengthening (TR90), maximal velocity of shortening/relengthening (±dL/ dt), and intracellular Ca2+ decay (&tgr;). HG myocytes exhibited normal PS; decreased ±dL/ dt; and prolonged TPS, TR90, and &tgr;. Interestingly, the HG-induced abnormalities were prevented with the AT1 blocker L-158,809 (10 to 1000 nmol/L) but not the Janus kinase-2 (JAK2) inhibitor AG-490 (10 to 100 &mgr;mol/L). The only effect of AT1 blockade on NG myocytes was enhanced PS at 1000 nmol/L. AT1 antagonist-elicited cardiac protection against HG was nullified by the NADPH oxidase activator sodium dodecyl sulfate (80 &mgr;mol/L) and mimicked by the NADPH oxidase inhibitors diphenyleneiodonium (10 &mgr;mol/L) or apocynin (100 &mgr;mol/L). Western blot analysis confirmed that the protein abundance of NADPH oxidase subunit p47phox and the AT1 but not the AT2 receptor was enhanced in HG myocytes. In addition, the HG-induced increase of p47phox was prevented by L-158,809. Enhanced reactive oxygen species production observed in HG myocytes was prevented by AT1 blockade or NADPH oxidase inhibition. Collectively, our data suggest that local Ang II, acting via AT1 receptor–mediated NADPH oxidase activation, is involved in hyperglycemia-induced cardiomyocyte dysfunction, which might play a role in diabetic cardiomyopathy.

Molecular evolution of the reactive oxygen-generating NADPH oxidase (Nox/Duox) family of enzymes

BackgroundNADPH-oxidases (Nox) and the related Dual oxidases (Duox) play varied biological and pathological roles via regulated generation of reactive oxygen species (ROS). Members of the Nox/Duox family have been identified in a wide variety of organisms, including mammals, nematodes, fruit fly, green plants, fungi, and slime molds; however, little is known about the molecular evolutionary history of these enzymes.ResultsWe assembled and analyzed the deduced amino acid sequences of 101 Nox/Duox orthologs from 25 species, including vertebrates, urochordates, echinoderms, insects, nematodes, fungi, slime mold amoeba, alga and plants. In contrast to ROS defense enzymes, such as superoxide dismutase and catalase that are present in prokaryotes, ROS-generating Nox/Duox orthologs only appeared later in evolution. Molecular taxonomy revealed seven distinct subfamilies of Noxes and Duoxes. The calcium-regulated orthologs representing 4 subfamilies diverged early and are the most widely distributed in biology. Subunit-regulated Noxes represent a second major subdivision, and appeared first in fungi and amoeba. Nox5 was lost in rodents, and Nox3, which functions in the inner ear in gravity perception, emerged the most recently, corresponding to full-time adaptation of vertebrates to land. The sea urchin Strongylocentrotus purpuratus possesses the earliest Nox2 co-ortholog of vertebrate Nox1, 2, and 3, while Nox4 first appeared somewhat later in urochordates. Comparison of evolutionary substitution rates demonstrates that Nox2, the regulatory subunits p47phox and p67phox, and Duox are more stringently conserved in vertebrates than other Noxes and Nox regulatory subunits. Amino acid sequence comparisons identified key catalytic or regulatory regions, as 68 residues were highly conserved among all Nox/Duox orthologs, and 14 of these were identical with those mutated in Nox2 in variants of X-linked chronic granulomatous disease. In addition to canonical motifs, the B-loop, TM6-FAD, VXGPFG-motif, and extreme C-terminal regions were identified as important for Nox activity, as verified by mutational analysis. The presence of these non-canonical, but highly conserved regions suggests that all Nox/Duox may possess a common biological function remained in a long history of Nox/Duox evolution.ConclusionThis report provides the first comprehensive analysis of the evolution and conserved functions of Nox and Duox family members, including identification of conserved amino acid residues. These results provide a guide for future structure-function studies and for understanding the evolution of biological functions of these enzymes.

Synaptic localization of a functional NADPH oxidase in the mouse hippocampus.

Superoxide has been shown to be critical for hippocampal long-term potentiation (LTP) and hippocampus-dependent memory function. A possible source for the generation of superoxide during these processes is NADPH oxidase. The active oxidase consists of two membrane proteins, gp91phox and p22phox, and four cytosolic proteins, p40phox, p47phox, p67phox, and Rac. Upon stimulation, the cytosolic proteins translocate to the membrane to form a complex with the membrane components, which results in production of superoxide. Here, we determined the presence, localization, and functionality of a NADPH oxidase in mouse hippocampus by examining the NADPH oxidase proteins as well as the production of superoxide. All of the NADPH oxidase proteins were present in hippocampal homogenates and enriched in synaptoneurosome preparations. Immunocytochemical analysis of cultured hippocampal neurons indicated that all NADPH oxidase proteins were localized in neuronal cell bodies as well as dendrites. Furthermore, double labeling analysis using antibodies to p67phox and the presynaptic marker synaptophysin suggest a close association of the NADPH oxidase subunits with synaptic sites. Finally, stimulation of hippocampal slices with phorbol esters triggered translocation of the cytoplasmic NADPH oxidase proteins to the membrane and an increase in superoxide production that was blocked by inhibitors of NADPH oxidase. Taken together, our data suggest that NADPH oxidase is present in mouse hippocampus and might be the source of superoxide production required for LTP and memory function.